Human leishmaniasis has been rated as the second target next to malaria among the six major diseases identified by the WHO for intensive research and control efforts (1). Leishmania, a trypanosomatid parasite caused a wide spectrum of infection ranging from self-curing ulcer to often-fatal visceral diseases. Further, the disease has been recognized as an opportunistic infection in immunocompromised individuals particularly in patients infected with HIV (2). There is no vaccine in routine use and chemotherapy still relies on antimonial-based drugs, first used in early 20th century. The pentavalent antimonials (SbV) drugs Sodium stibogluconate (SAG) and N-methylglucamine antimoniate are the only anti leishmanials chemotherapeutic compounds with a certain degree of efficacy and safety. Sodium stibogluconate (Stibanate) first became available in 1945 (3). Two formulation of pentavalent antimonials are available, sodium stibogluconate solution (Pentosam, Wellcome Foundation, UK) containing 100 mg SbV/ml and meglumine asciculat solution (Glucantine, Rhone Poulence, France) containing 85 mg SbV/ml, and former is used in Indian subcontinent. Currently recommended dose of SbV is 20 mg/kg/day (MKD) for 30 days (4). The drug can be used via intramuscular or intravenous route. Response to relatively small daily dose (600 mg. Max.) for short duration (6-10 days) of SbV had been excellent until the early 1980 (5), when reports of treatment failure appeared, and modifications for SbV treatment were suggested to overcome the drug failure (6). The WHO revised its recommendations twice, resulting in an increase in the daily dose (from 10-20 mg/kg) and duration (from 6-10 days to 20-40 days) (7, 8). However, non-responsiveness to SAG is on increase especially in epidemic areas of visceral Leishmaniasis (9).
Leishmania protein are generally insoluble in nature and tend to form aggregate i.e. inclusion bodies upon expression in prokaryotic hosts e.g. Adenylate kinase 2 of L. donovani (10), Methionine adenosyl transferase (MAT 2) of L. donovani (11), Cysteine protease type A & B (CPA & CPB) genes of L. infantum (12), Glucose regulating protein 94 (GRP 94) of L. infantum (13), Myristoyl-CoA N Myristoyl transferase of L. major (14). To get active protein from inclusion bodies is a tedious process and requires lot of laboratory work and time. Some researchers has worked upon it and tried many conditions to get active protein in the soluble fraction but the yield is too low to work upon this e.g. L. mexicana aminotransferase was expressed to 1 mg./l bacterial culture (15), L. donovani recombinant chitinase Ld CHT1 (16), Ornithine decarboxylase (ODC) was expressed at 0.2% of the soluble protein in E. coli. While Thymidylate synthase dihydrofolate reductase was expressed at a level of 2% of the soluble protein in E. coli. (17). The soluble functionally active enzyme must be substrate specific and should have other kinetic parameters in accordance with the natural enzyme. Therefore, a process needs to be developed for large-scale heterologous expression and purification of functionally active leishmanial proteins. Also, there is an increasing failure of insecticides to control vector. This has led to research on the basic studies to evaluate the significant differences between host and parasite, which leads to development of logical approaches towards new chemotherapeutic agents and vaccines. To validate new targets as well as new molecules active against the parasite requires lot of native target enzyme thereof and it is therefore necessary to get the target enzyme/protein in large amount by developing a heterologus expression system.
As parasites, the trypanosomatids are inevitably exposed to various reactive oxygen species, such as superoxide radicals, hydrogen peroxide and myeloperoxidase products generated during the host defense reaction. However there ability to cope with such oxidative stress appears to be surprisingly weak. They lack both catalase & peroxidase and totally dependent upon unique Trypanothione reductase redox system to overcome this stress. Trypanothione Reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in the leishmanias and trypanosomatids (18). The uniqueness of this cascade of oxidoreductase offers an opportunity to inhibit the parasite metabolic pathway without causing adverse effects in the host organism therefore make this enzyme an attractive target site for the development of the antileishmanials.
The importance of trypanothione and trypanothione reductase in defending trypanosomatids against nitrosative and oxidative stress was established by disabling the function of trypanothione reductase gene by gene disruption. A double knock out of TR gene in Leishmania species could not be achieved which indicated that null mutants are not viable hence proved importance of the gene in parasite survival (Dumas, C., Quellette, M., Tovar, J., Cunningham, M. L., Fairlamb, A. H., Tamar, S., Olivier, M. and Papadopoulou, B. (1997), EMBO J., 16, 2590-2598). Mutants in which the TR activity had been reduced to half that of wild type by disrupting one member of allelic pair were not able to survive in macrophages that were capable of respiratory burst.
In another approach which relies on the fact that the active enzyme is dimeric in nature an expression vector was constructed bearing inactive mutant of T. cruzi TR gene. This expression construct was heterologously expressed in L. donovani. In the resultant recombinants, a large proportion of the TR consisted of inactive heterodimer with the result that TR activity was reduced to approximately 15% of normal cells (Tovar, J., Cunningham, M. L., Smith, A. C., Croft, S. L. and Fairlamb, A. H. (1998) PNAS, 95, 5311-16). These recombinant L. donovani cells expressing very less active TR were incapable of surviving in IFN-gamma activated macrophages. This further confirms that parasite with much less TR activity than wild type were more vulnerable to oxidative and nitrosative stress”.
Heterologous expression means the expression of a gene from one organism into another organism. In the paper by Tovar et al, 1998, an inactive mutant of T. cruzi TR was expressed in L. donovani cells resulting recombinant Leishmania cells expressing inactive TR. While, we have expressed the Leishmanial TR gene in E. coli cells resulting recombinant bacterial cells expressing active Leishmanial TR which was not done before. These recombinant bacterial cells were used to purify Active Leishmanial enzyme. Though in both cases the expression of TR was heterologous but in first case it was expression of inactive mutant TR of T. cruzi in L. donovani while in second it was expression of active L. donovani TR in E. coli cells.
The main objective of this patent is to get L. donovani TR in very high yield and at very low cost. This was achieved by expressing L. donovani TR in E. coli bacterial cells. The enzyme can be purified from the parasite itself but the process is very expensive, cumbersome therefore time consuming. Moreover the yield was very low [Cunningham, M. L. and Fairlamb, A. H. (1995) Eur. J. Biochem., 230, 460-468]. To grow one liter of promastigotes of L. donovani in liquid 199 medium involves an expenditure of approximately $57.00 which will generate approximately 2.5 g parasite while to grow 1 L bacterial culture that comes only $1.69 generating 12.5 g cells. Further purification steps to process 1 L parasite culture will cost $ 1011.8 resulting in total yield of only 0.47 mg protein while purification from bacterial 1 L culture will cost only 131$ with a total yield of approximately 16 mg protein (Table).
The expression vectors used in the present study have far more advantage in obtaining the desired results as revealed by the present study. In addition to pET41a we tried four more expression vectors but the yield of active protein was very-very less. Almost all of the expressed protein was inactive in the form of inclusion bodies. The best yield of the active recombinant Leishmanial TR was observed with pEt41a vector expressing in E. coli cells strain BL21.
So far Trypanothione reductase has been isolated from C. asciculate itself (19) and from Trypanosoma cruzi (2.2 mg. TR/33 g wet weight of cultured epimastigotes) (20). Briefly, 33.4 g cells were suspended in 100 ml of buffer A (50 mM Potassium phosphate, 1 mM EDTA, pH7.0 at 25° C.) in presence of 1 mM digitonine. After 10 min. of gentle stirring the suspension was freeze thawed for 2 cycles and centrifuged for 30 min at gravitational force, 4000 g and temperature 4° C. The supernatant was combined with 50 ml of 2′5′ ADP sepharose pre-equilibrated with buffer A. The suspension was shaken for 2 h and transferred to a chromatography column. Column was washed first with 150 ml of buffer A (at 4° C.) followed by 60 ml of buffer B (RT) TR was eluted using 75 ml of 0.3 mM NADPH in buffer B. Active fractions were combined to give TR in 2 electron-reduced form (EH2) which is susceptible to nonspecific auto oxidation. TR (EH2) was reduced by addition of TS2 and GSSG to a final conc. Of 1.6 μM and 1 mM respectively. Reaction was allowed to proceed for 30 min. at 25° C. resulting in fraction 2. Fraction 2 was applied on to DEAE sephadex A-50 column pre-equilibrated with buffer B. Column washed with buffer B and yellow band of TR was eluted with 0.5M KCl. Active fractions were combined. TR was precipitated with slow addition of solid ammonium sulfate to 60% saturation. After 24 h of precipitation at 4° C. the sample was centrifuged at gravitational force, 6000 g for 10 min at 4° C. temperature. The yellow precipitate was washed thrice with 45% ammonium sulfate in buffer A resulting in 95% pure trypanothione reductase.
In 1989, first time, the cloned TR gene from Trypanosoma congolens had been expressed in E. coli, where the yield was only 1% of the total soluble fraction of the bacterial cells (17). The cell pellet obtained from 1 L of culture of induced cells were suspended in 25 ml buffer A (20 mM phosphate buffer pH7.2, 5 mM BME, 1 mM EDTA), sonicated six times for 1 min each and centrifuged. Nucleic acids were removed by addition of protamine sulphate to 0.4% concentration. The cloudy solution was then brought to 40% saturation with ammonium sulfate. Precipitated proteins and nucleic acid were removed by centrifugation and TR in supernatant was precipitated with 60% ammonium sulphate. The resulting pellet of TR was dissolved in buffer B (20 mM Tris, 5 mM BME, 1 mM EDTA pH7.2) and dialyzed extensively.
The dialyzed solution was applied to DEAE sephacel (pre-equilibrated) column. The TR was eluted at 0.1M KCl as yellow protein, which was dialyzed against buffer A. The yellow protein solution was applied on to 2′5′ ADP sepharose pre-equilibrated column. The column was washed with 100 ml of buffer A and pure TR was eluted with 5 mM NADP+ in buffer A. The enzyme was dialyzed against buffer A and stored at 4° C. after concentration. Thus, the purification process was quite lengthy and involves several steps of purification.
TR from Trypanosoma cruzi was cloned and expressed in E. coli (21). However, all the steps for the purification of recombinant TR were almost same as described for T. congolens (17) with a yield of approximately 6 mg/l bacterial culture.
TR from L. donovani Ethiopian strain has been cloned, over-expressed in parasite itself (22). However, purification of the enzyme from overexpressing parasite resulted in very poor yield to work upon (23). No reports are available for heterologous expression of Leishmanial trypanothione reductase in prokaryotic system.
We have presented a comparative data in table 1 which shows the present process is far more advantages than the earlier known methods. Further table 2 shows another comparison cost effectiveness of the present method than the known methods.
TABLE 1Comparison of the present invention with the prior artSourceVectorHost cellsPurification proceduresYieldT. congolens (TR)PCGTR-2E. coli1. Salt precipitation - 40-60%,3.2 mg/L(ref. 17)(SG5)2. Anion exchange chromatography - DEAE sephacel,bacterial3. Affinity chromatography- 2′5′ADP-sepharose,cultureT. cruzi (TR)pIBITczTRE. coli 1. Salt precipitation - 40-60%,6 mg/L(ref. 21)2. Anion exchange chromatography-DEAE sephacel,bacterial3. Affinity chromatography- 2′5′ADP-sepharose,cultureC. fasciculateFromFrom1. Salt precipitation - 40-60%,0.49 mg/L(Ref. 19)parasiteparasite2. Anion exchange chromatography - DEAE sephacel,of parasite3. Hydroxyapitite chromatographyculture4. Affinity chromatography- 2′5′ADP-sepharose,5. UltragelL. donovani (Dd8pET-41aE. coli-Single step - Glutathione--sepharose affinity16 mg/lstrain) (PresentBL21chromatographybacterialinvention)(DE3)culture
Comparison of the cost in US$ required to purify 1 mg of LDTR enzyme from 1L. donovani promastigotes and recombinant E. coli cells (Table 2)
TABLE 2Cost (ParasiteCost (BacterialS. No.Steps involved in purification of TRL. donovani)US$E. coli) US$1.Culture of 1 L57.001.692.Purification ProcessThree step (two affinity &Single(affinityone size chromatography)chromatographycost = 1011.80Cost = 131.003.Time (from culture to purification)12 days2.5 days4.Yield of Enzyme0.4716 mg5.Cost/mg protein1239.008.29